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Original Study

Utility of Antimicrobial Susceptibility Testing in Trichomonas vaginalis–Infected Women With Clinical Treatment Failure

Bosserman, Elizabeth A. MPH*†; Helms, Donna J. MPH; Mosure, Debra J. PhD; Secor, W. Evan PhD; Workowski, Kimberly A. MD†‡

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doi: 10.1097/OLQ.0b013e318224db39
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Trichomonas vaginalis is the causative agent of trichomoniasis, a sexually transmitted infection affecting approximately 7 million Americans yearly.1–3 In the United States, the estimated prevalence of trichomoniasis in all women of reproductive age is 3.1%, with a prevalence of 13.5% in non-Hispanic black women and 1.2% in non-Hispanic white women.4 Although both men and women can be infected, women are more likely to experience clinical manifestations, including green or yellow vaginal discharge, vaginal odor, dysuria, irritation, itching, lower abdominal pain, strawberry cervix, and inflammation.3,5 Infection with T. vaginalis in women has been associated with a number of sequelae, including increased risk of HIV acquisition, premature labor, and low birth weight infants.5–8

Treatment options for trichomonas infection are limited and include 2 oral 5-nitromidazoles, metronidazole and tinidazole. The oral bioavailability of metronidazole is greater than 90%; however, the concentration of drug that reaches the urogenital area is unknown.9,10 Tinidazole has a biologic half-life twice as long as that of metronidazole, better in vitro activity against trichomonads, and is well tolerated.9,11–14 Both of these nitroimidazoles are approved for the treatment of trichomoniasis in the United States by the Food and Drug Administration and recommended treatment includes a single 2 g oral dose of metronidazole or tinidazole.6 The cure rate for trichomoniasis using the recommended nitroimidazole regimens is estimated to be 86% to 100%.6 If the single-dose nitroimidazole regimen is unsuccessful, metronidazole 500 mg twice a day for 7 days is recommended. For those persons failing this regimen, tinidazole or metronidazole at 2 g orally for 5 days is recommended.6 If these regimens are not effective, further management should be discussed with a specialist.

For women whose infections are not eradicated after the standard regimens, antimicrobial resistance should be considered. The first report of metronidazole treatment failure in a woman with trichomoniasis occurred within 2 years of its introduction in 1960, which may suggest that acquired resistance of the organism is not the only factor in drug resistance. A degree of drug tolerance among trichomonads, insufficient absorption of metronidazole, or inadequate transport of the drug to the site of infect may affect treatment outcomes.15 Since trichomoniasis is not routinely reported to health departments, the true prevalence of clinical treatment failure due to antimicrobial resistance is unknown. Current estimates of nitroimidazole resistance range between 2% and 6% for women attending urban gynecological clinics, with an occurrence of as high as 10%.12,16–18

For individuals whose infections are not cured following standard therapy or who display hypersensitivity to nitroimidazole drugs, intravaginal treatments with furazolidone, povidone-iodine, and paromomycin sulfate have shown some success.19–22 Although these drugs remain an option for women whose infections are not successfully eradicated using the recommended oral nitroimidazole regimens, they have high failure rates. A recent study evaluating treatment options for women with hypersensitivity to nitroimidazoles found that desensitization with nitroimidazoles was more effective than intravaginal treatments in eradicating trichomonas infection.21

The Division of STD Prevention and the Division of Parasitic Diseases and Malaria at the Centers for Disease Control and Prevention (CDC) provide consultation to health care providers and susceptibility testing on submitted isolates from women who have experienced clinical failure to standard treatment.6 After in vitro nitroimidazole susceptibility testing and review of the treatment history, alternative treatment options are discussed with the clinician. For many women with metronidazole-resistant isolates, tinidazole administered for a prolonged duration at a higher daily dose results in a cure.23–25 However, because both drugs are nitroimidazoles with the same mode of action, cross resistance can occur.11,13,26

In the same way, the usefulness of alternative recommendations for women with nitroimidazole hypersensitivity was recently assessed.21 We sought to evaluate alternative treatment regimens for women with clinically resistant T. vaginalis infections. In this report, we describe alternative treatment options for women with repeated clinical nitroimidazole treatment failure, document in vitro susceptibility testing findings, and assess the utility of susceptibility testing for patient care.

MATERIALS AND METHODS

Laboratory Methods

Health care providers who contacted the CDC's Division of STD Prevention or Division of Parasitic Diseases and Malaria (DPDM) seeking consultation for trichomonas infection in women with reported clinical treatment failure were provided with a culture media for the collection of vaginal specimens to submit to the CDC. Clinicians were asked to provide information on patient age, weight, race, current and previous symptoms, treatment history, sexual partner treatment, sexual intercourse since previous treatment, and provider assessment of treatment adherence.

Providers obtained vaginal specimens and inoculated a tube of modified Diamond's medium containing agar and incubated it at 37°C for 24 hours.27 Providers were asked to prepare a wet mount and examine the specimen by microscopy at a magnification of 100× to confirm the presence of viable, motile trichomonads. If trichomonads were present, a subculture was prepared in a second tube and both tubes were sent to DPDM by overnight courier. If trichomonads were not present, the specimen was incubated for an additional 24 to 48 hours. If motile trichomonads could then be detected on a wet mount, the specimen and subcultures were sent to the DPDM laboratory.

When the isolates arrived at the DPDM laboratory, they were transferred into a fresh tube of Diamond's media and passaged until axenic T. vaginalis cultures were obtained. Penicillin, streptomycin, normocin, and fungizone were used to inhibit the growth of contaminating organisms. Antimicrobial susceptibilities to metronidazole and tinidazole for each isolate were measured under both aerobic and anaerobic conditions using a technique developed by Meingassner and Thurner.28 Dimethyl sulfoxide (DMSO) was used to solubilize the nitroimidazoles and as a control for parasite viability. Parasites were cultured at 104 cells per well in round bottomed microtiter plates in serial 2-fold concentration dilutions (400 to 0.1 μg/mL) of each compound. Plates were incubated at 37°C for 48 hours and read on an inverted microscope. Reference resistant and susceptible control strains were used to monitor the validity of each assay. Susceptibility testing results were reported as the minimum lethal concentration (MLC) or the lowest concentration of drug at which no viable trichomonads are observed.16,24 Resistance testing was conducted in triplicate for each drug concentration, and the assays were repeated on 2 separate dates to ensure reproducibility of the results.

In vitro susceptibility to metronidazole was defined as an aerobic MLC of less than 50 μg/mL. Isolates with MLCs of 50 to 100 μg/mL were classified as minimally resistant, isolates with MLCs of 200 μg/mL were considered moderately resistant, and isolates with MLCs of 400 μg/mL or greater were classified as highly resistant.11 Tinidazole resistance levels were assigned using the same MLC values as metronidazole.

Clinical Consultation

Based on the clinician-provided patient history and the in vitro susceptibility testing results, treatment options were discussed with the health care provider by an infectious diseases specialist knowledgeable in the management of women with clinical treatment failure (KW). For women whose isolates were susceptible to metronidazole, the recommended treatment regimen was 3 g of oral metronidazole daily for 14 days. For women with minimally resistant isolates, the recommended regimen was 2 g of oral tinidazole daily for 5 to 7 days. For women with moderately and highly resistant isolates, the recommended regimen was 1 g of oral tinidazole 3 times daily and 500 mg of tinidazole administered intravaginally twice daily for 14 days.24 An additional option for women with highly resistant isolates was treatment with vaginal furazolidone twice daily for 10 to 14 days alone or in combination with high-dose tinidazole.

Data Collection and Analysis

Following provision of a treatment recommendation based on in vitro results and patient history, health care providers were contacted to ascertain what drug regimen they used and whether the patient was successfully cured. Follow-up data were solicited from providers who contacted the CDC's Division of STD Prevention or DPDM for consultation between January 2002 and January 2008. During that period, there were approximately 500 kit requests for nitroimidazole susceptibility testing in response to clinical treatment failure. From these requests, approximately 300 samples were sent for susceptibility testing. Viable isolates were obtained from 175 of these samples and were successfully cultured and assayed. The remaining samples were nonviable, contained no trichomonads, or were too heavily contaminated to undergo susceptibility testing.

In March 2004, additional information was requested following the provision of the alternative treatment recommendations. This included the actual treatment regimen administered, occurrence of adverse events, and treatment outcomes. If the provider did not submit follow-up information within 4 months postconsultation, telephone contact was initiated. Follow-up information from providers who requested consultation before March 2004 was obtained retrospectively by telephone or fax.

The outcome of treatment was determined based on both clinical improvement and available microbiologic evaluation. Confirmed microbiologic cure was defined as a negative trichomonas culture 1 to 4 weeks after treatment. A negative wet mount, in addition to the complete resolution of signs and symptoms, was defined as a suspected microbiologic cure. If no test of cure was performed, the treatment outcome was based on the provider's clinical impression. A clinical cure was defined as complete resolution of signs and symptoms of trichomoniasis. A treatment failure was defined as women whose symptoms were not resolved, those whose infection relapsed in the absence of treatment noncompliance or reinfection, or those with a positive wet mount or culture following treatment.

RESULTS

Initial Consultation

The 175 women whose isolates were successfully tested for drug resistance had a median age of 36.5 years (range: 15–67 years). Forty-two percent of the women were black, 37% white, and 6% “other.” Race was not available for 26 (15%) women. The median weight was 170 pounds (range: 98–360 pounds). Data on weight were not available for 81 (46%) of the women.

Seventy-five percent of the 175 women had failed 3 or more previous treatments for trichomoniasis before the initial consultation. For 51 women (29%), the highest total dose of metronidazole given in a treatment regimen was between 10 and 14 g. Noncompliance with drug treatment regimens was suspected in 7 (4%) of the 175 women.

In vitro, 115 of the 175 isolates demonstrated resistance to metronidazole (aerobic MLC of ≥50 μg/mL): 35 (20%) isolates were minimally resistant, 24 (14%) isolates were moderately resistant, and 56 (32%) were highly resistant (Table 1). The remaining 60 (34%) trichomonas isolates had MLCs <50 μg/mL. For all isolates resistant to metronidazole, the in vitro resistance level for tinidazole was similar or lower. Sixty-one of 175 isolates demonstrated in vitro tinidazole resistance: 28 (16%) were minimally resistant, 13 (7%) were moderately resistant, and 20 (11%) were highly resistant. The remaining 114 isolates (65%) had MLCs <50 μg/mL (Table 1). All of the 61 isolates resistant to tinidazole were also resistant to metronidazole.

TABLE 1
TABLE 1:
Distribution of Clinically Resistant Isolates by In Vitro Maximum Lethal Concentration (MLC) Levels for Women With Follow-Up Results

Response to Treatment

Follow-up information was available for 72 (41%) of the 175 women for whom in vitro nitroimidazole susceptibility data were available. Comparison of the population of women who had follow-up data available with those without available follow-up information indicated no significant differences across demographic variables, treatment histories, patient weight, or isolate in vitro susceptibilities (data not shown).

During the first round of treatment following provision of a treatment regimen based on in vitro results and treatment history, 52 (72%) of the 72 women with follow-up information were treated with a nitroimidazole (metronidazole or tinidazole), 18 (25%) women were treated with intravaginal furazolidone, and 2 (3%) women received intravaginal paromomycin. Not all women received treatments in accordance with the recommended alternative recommendation. Of the women receiving the recommended nitroimidazole dose or higher, 30 (83%) of 36 were cured compared with 8 (57%) of 14 women who received a regimen administered at a lower dose than recommended (Table 2).

TABLE 2
TABLE 2:
Treatment Outcomes by In Vitro Metronidazole Resistance Level and Nitroimidazole Received, Initial Alternative Regimen Following Susceptibility Testing

Intravaginal furazolidone, which was recommended for some women whose isolates displayed high in vitro resistance, eradicated infection in only 6 (33%) of the 18 women. Intravaginal paromomycin was successful for the 2 women who received it: one whose isolate was minimally resistant and other who was infected with a highly resistant isolate.

The 25 women whose infections were not cured by the first treatment course following susceptibility testing received additional courses of treatment. An additional 8 women were cured with a second treatment course, 2 women during the third course of treatment, and 2 women during the fourth course of treatment. In total, 59 (82%) of the 72 women cleared their infections after one or more treatment courses: 22 demonstrated a microbiologic cure, 33 had a suspected microbiologic cure, and 4 were clinically cured. The 13 (18%) remaining women were lost to follow-up. Trichomonas infections were cured with an average of 1.3 (range: 1–4) treatments following isolate susceptibility testing.

Between 2002 and 2008, 20 viable isolates demonstrated high resistance in vitro to both metronidazole and tinidazole. Follow-up information was available for 9 (45%) of these 20 women. Tinidazole, 3 g orally and 1 g intravaginally, successfully eradicated infection in 2 women; intravaginal paromomycin treatment eradicated infection in 1 woman; and 6 women received intravaginal furazolidone, but only 3 were successfully cured. The 3 women whose infections were not successfully eradicated by initial furazolidone treatment received additional treatments. Two women received tinidazole (2 g per day for 5 days and 3 g per day for 14 days) and were cured; intravaginal furazolidone failed to cure the third woman on 3 separate occasions.

DISCUSSION

In recent years, there have been increasing reports of clinical treatment failure in women with vaginal trichomoniasis.12,16,17 Although this may reflect an increase in the number of resistant T. vaginalis infections, it is also likely a reflection of growing numbers of providers aware of nitroimidazole resistance in trichomoniasis. In an effort to assess the impact of drug resistance testing and recommendation service on clinically resistant T. vaginalis infections, we followed up with clinicians to whom alternate treatment regimens were provided.

Overall, the majority of women with clinical treatment failure had their infections eradicated (82%). Eradication rates were higher for women who were treated with nitroimidazoles in accordance with or beyond the alternative recommendation provided after in vitro testing compared with those who did not follow the recommended alternative treatment regimen.

One-third of the submitted isolates in women with clinical treatment failure were susceptible to metronidazole in this in vitro assay. While most of the patients suspected of noncompliance with previous treatments were in this group, we were unable to explain why apparent clinical resistance occurred in women infected with isolates that had in vitro MLCs typically associated with susceptibility to metronidazole.29 Neither patient weight nor number of previous treatments could explain this finding. However, the rapid appearance of patients who failed to eradicate trichomoniasis before use of metronidazole became widespread (i.e., before it is likely that resistance could be induced) suggests that clinical treatment failure may be due to inherent drug tolerance in some isolates or that the bioavailability of drug in some women may be reduced.15

A majority of women (65%) who exhibited clinical treatment failure did not have tinidazole-resistant isolates by in vitro susceptibility testing. The same cut-offs for antimicrobial susceptibility were used for both nitroimidazoles. However, because the in vitro tinidazole MLC that correlates with clinical treatment failure has not been validated, the percentage of isolates classified as tinidazole resistant may be inaccurate. Nevertheless, with greater efficacy compared with metronidazole and reduced side effects, tinidazole is recommended for treating T. vaginalis infections that demonstrate clinical treatment failure; however, tinidazole can be more expensive.11

For isolates with high in vitro resistance or women with severe allergic reactions (e.g., Stevens-Johnson syndrome) to the 5-nitroimidazoles, treatment options are limited. Vaginal furazolidone treatment resulted in a cure rate of only 33%. Paromomycin, which is also administered intravaginally, demonstrated 100% efficacy in the 2 patients who received this drug. It has also been effective to treat other patients with metronidazole-resistant infections, although with a risk of causing vaginal ulcerations.22

Because at least 82% of the women who initially presented with resistant trichomoniasis were eventually cured, we believe that the antimicrobial susceptibility testing and treatment recommendations play a useful role in management of women with clinical treatment failure.

This analysis has some limitations. Women included in this case series are not representative of all women with trichomoniasis and clinical treatment failure, as they were obtained through a passive consultation service. In addition, the geographic distribution of women included in this study was not uniform across the United States. However, it is not clear how active isolate acquisition or a more uniform geographic distribution of isolates would have changed our findings. Also, it is possible, if not likely, that a number of the women failed to return for their subsequent appointments because they had resolved their symptoms (i.e., had experienced clinical cure) which may have resulted in the reported cure rates being lower than true rate.

Another limitation was the low percentage of successfully cultured isolates; 175 (58%) of 300 specimens submitted were able to be grown in vitro. We theorize that some of these women were not infected with T. vaginalis although they demonstrated symptoms consistent with trichomoniasis. As a result, the reason for their clinical treatment failure was failure to treat the underlying cause of their symptoms. For other women, coinfections with other bacterial or fungal diseases prevented successful growth of the trichomonads. In those instances, women should be treated for their coinfections and then recultured. We did not have information on concomitant bacterial vaginosis, which may have affect treatment results. For other isolates, the trichomonads may not have survived the shipping process, as proper shipping and handling procedures are necessary when submitting a specimen for nitroimidazole antimicrobial resistance testing.

Additional research to evaluate the prevalence of nitroimidazole resistant trichomoniasis is needed. Limited point prevalence studies in the United States suggest resistance levels between 2% and 6%.12,16,18 Although most isolates from women with trichomoniasis treatment failures demonstrate in vitro resistance, there are also other possible, as yet undefined, reasons for treatment failure. For women with clinical treatment failure, investigating achievable intravaginal nitroimidazole concentrations may provide additional insights. Due to the limited options of antimicrobial regimens recommended for trichomoniasis treatment in persons with clinical treatment failure or nitroimidazole hypersensitivity, additional research is needed to investigate alternative therapeutic options for treatment.

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